Androgen receptor modulator for the treatment of prostate cancer and androgen receptor-associated diseases

ABSTRACT

Described herein, inter alia, are compounds useful for the prevention or treatment of hyperproliferative diseases or disorders.

FIELD OF THE INVENTION

The present invention relates to hydantoins, thiohydantoins,dithiohydantoins, hydantoinimines and thiohydantoinimines compounds,methods of using such compounds in the treatment of androgenreceptor-associated conditions, such as age-related diseases, forexample, prostate cancer, and to pharmaceutical compositions containingsuch compounds.

BACKGROUND OF THE INVENTION

Prostate cancer is the most common incidence of cancer and the secondleading cause of cancer death in Western men. When the cancer isconfined locally, the disease can be cured by surgery or radiation.However, 30% of such cancer relapses with distant metastatic disease andothers have advanced disease at diagnoses. Advanced disease is treatedby castration and/or administration of anti-androgens, the so-calledandrogen deprivation therapy. Castration lowers the circulating levelsof androgens and reduces the activity of androgen receptor (AR).Administration of anti-androgens blocks AR function by competing awayandrogen binding and therefore reduces the AR activity. Althoughinitially effective, these treatments quickly fail and the cancerbecomes hormone refractory.

Recently, overexpression of AR has been identified and validated as acause of hormone refractory prostate cancer (Nat. Med, 2004, 10, 33-39).Overexpression of AR is sufficient to cause progression from hormonesensitive to hormone refractory prostate cancer, suggesting that betterAR inhibitors than the current drugs can slow the progression ofprostate cancer. It was demonstrated that AR and its ligand binding arenecessary for growth of hormone refractory prostate cancer, indicatingthat AR is still a target for this disease. It was also demonstratedthat overexpression of AR converts anti-androgens from antagonists toagonists in hormone refractory prostate cancer (an AR antagonistinhibits AR activity and an AR agonist stimulates AR activity). Datafrom this work explain why castration and anti-androgens fail to preventprostate cancer progression and reveals un-recognized properties ofhormone refractory prostate cancer.

Bicalutamide (Brand name: Casodex) is the most commonly usedanti-androgen. While it has inhibitory effect on AR in hormone sensitiveprostate cancer, it fails to suppress AR when the cancer becomes hormonerefractory. Two weaknesses of current antiandrogens are blamed for thefailure to prevent prostate cancer progression from hormone sensitivestage to hormone refractory disease and to effectively treat hormonerefractory prostate cancer. One is their weak antagonistic activitiesand the other is their strong agonistic activities when AR isoverexpressed in hormone refractory prostate cancer. Therefore, betterAR inhibitors with more potent antagonistic activities and minimalagonistic activities are needed to delay disease progression and totreat the fatal hormone refractory prostate cancer.

Nonsteroidal anti-androgens, have been preferred over steroidalcompounds for prostate cancer because they are more selective and havefewer side effects. A wide variety of such compounds were described inU.S. Pat. Nos. 4,097,578, 5,411,981, and 5,705,654, U.S. publishedapplications 2004/0009969 and 2007/0004753, and PCT internationalapplications published as WO 97/00071, WO 00/17163 and WO 06/124118.

Accordingly, identification of compounds which have high potency toantagonize the androgen activity, and which have minimal agonisticactivity would overcome the hormone refractory prostate cancer (HRPC)and avoid or slowdown the progression of hormone sensitive prostatecancer (HSPC). There is a need in the art for the identification ofselective modulators of the androgen receptor, such as modulators whichare non-steroidal, non-toxic, and tissue selective.

SUMMARY OF THE INVENTION

A series of compounds that modulate the function of the nuclear hormonereceptors, especially the androgen receptor are presented. Thesecompounds can cause disappearance of prostate cancer cells and tumors.

In an embodiment, a compound is according to formula II.

Het represents a heterocyclic unit of 5 or 6 atoms. A and B areindependently selected from oxygen, sulfur, and N—R₉, with R₉ beingselected from hydrogen, aryl, substituted aryl, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,halogenated alkyl, halogenated alkenyl, halogenated alkynyl, arylalkyl,arylalkenyl, arylalkynyl, heterocyclic aromatic or non-aromatic,substituted heterocyclic aromatic or non-aromatic, cycloalkyl,substituted cycloalkyl, SO₂R₁₁, NR₁₁R₁₂, NR₁₂(CO)OR₁₁, NH(CO)NR₁₁R₁₂,NR₁₂(CO)R₁₁, O(CO)R₁₁, O(CO)OR₁₁, O(CS)R₁₁, NR₁₂(CS)R₁₁, NH(CS)NR₁₁R₁₂,or NR₁₂(CS)OR₁₁. R₁₁ and R₁₂ are independently selected from hydrogen,alkyl, substituted alkyl, alkenyl or substituted alkenyl, alkynyl orsubstituted alkynyl, aryl, substituted aryl, arylalkyl, arylalkenyl,arylalkynyl, heterocyclic aromatic or non-aromatic, or substitutedheterocyclic aromatic or non-aromatic. R₁ is selected from hydrogen,aryl, substituted aryl, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, halogenated alkyl, halogenatedalkenyl, halogenated alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heterocyclic aromatic or non-aromatic, substituted heterocyclic aromaticor non-aromatic, cycloalkyl, substituted cycloalkyl, SO₂R₁₁, NR₁₁R₁₂,NR₁₂(CO)OR₁₁, NH(CO)NR₁₁R₁₂, NR₁₂(CO)R₁₁, O(CO)R₁₁, O(CO)OR₁₁, O(CS)R₁₁,NR₁₂(CS)R₁₁, NH(CS)NR₁₁R₁₂, or NR₁₂(CS)OR₁₁. R₂ and R₃ are independentlyselected from hydrogen, aryl, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, halogenated alkyl,halogenated alkenyl, halogenated alkynyl, arylalkyl, arylalkenyl,arylalkynyl, heterocyclic aromatic or non-aromatic, substitutedheterocyclic aromatic or non-aromatic, cycloalkyl, or substitutedcycloalkyl, or, together with the carbon to which they are linked, forma cycle which can be cycloalkyl, substituted cycloalkyl, heterocyclicaromatic or non-aromatic, substituted heterocyclic aromatic ornon-aromatic.

R₁ and R₂ can be connected to form a cycle which can be heterocyclicaromatic or non aromatic, substituted heterocyclic aromatic or nonaromatic. R₁₁ and R₁₂ can be connected to form a cycle which can beheterocyclic aromatic or non-aromatic, substituted heterocyclicaromatic, cycloalkyl, or substituted cycloalkyl.

For example, the compound can be A51 or A52.

In an embodiment, a pharmaceutical composition includes atherapeutically effective amount of a compound according to Formula II,or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier, diluent, or adjuvant.

The pharmaceutical composition can include a solution ofdimethylsulfoxide, phosphate buffered saline solution, and water. Thepharmaceutical composition can include dimethylsulfoxide, acarboxymethylcellulose, a polysorbate, and water.

An embodiment of a method includes preventing or treating a disease ordisorder related to nuclear receptor activity.

A method for preventing or treating a hyperproliferative disorder, suchas hormone sensitive prostate cancer or hormone refractory prostatecancer, can include administering a compound according to Formula II, ora pharmaceutically acceptable-salt thereof, to a subject in need of suchprevention or treatment, thereby preventing or treating thehyperproliferative disorder. The compound can be administered at adosage in the range of from about 1 mg per kg body weight per day toabout 50 mg per kg body weight per day. The compound can beadministered, for example, by intravenous injection, by injection intotissue, intraperitoneally, orally, or nasally.

In an embodiment, the compound according to Formula II is an antagonistof a nuclear receptor or an antagonist of an androgen receptor.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar chart depicting the antagonist effect of compounds A51and A52 on HS cancer cells.

FIG. 2 is a bar chart depicting the antagonist effect of compounds A51and A52 on HS cancer cells.

FIG. 3 is a bar chart depicting the antagonist effect of compounds A51and A52 on HR cancer cells.

FIG. 4 is a graph depicting the pharmacokinetic behavior of compoundA52.

FIG. 5 is a graph depicting the effect of compound A52 onLnCaP-AR-overexpressed tumor size at 10 mg/kg.

FIG. 6 presents images depicting the disappearance of Luciferaseactivity after 17 days of treatment with compound A52.

DETAILED DESCRIPTION

Embodiments of the invention are discussed in detail below. Indescribing embodiments, specific terminology is employed for the sake ofclarity. However, the invention is not intended to be limited to thespecific terminology so selected. A person skilled in the relevant artwill recognize that other equivalent parts can be employed and othermethods developed without parting from the spirit and scope of theinvention. All references cited herein are incorporated by reference asif each had been individually incorporated.

The present invention relates to the compounds of formula II, methods ofusing such compounds as modulators of androgen receptors and topharmaceutical compositions containing such compounds and salts thereof.Compounds of formula II can be used to agonize or antagonize thefunction of the nuclear receptor. The compounds can be used toantagonize the androgen receptor. Use of the compounds is not limited toaffecting the androgen receptor, but can, for example, also be usefulfor the treatment of other diseases related to nuclear receptorfunction. Formula II can be represented as the structure

wherein, Het is a heterocyclic unit of 5 and 6 atoms. Preferredheterocyclic units are selected from compounds represented by thestructures

and the like. However, the invention is not intended to be limited tocompounds having these structures.

Herein, R₄, R₅, R₆, and R₇ are independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,arylalkyl, arylalkenyl, arylalkynyl, halogenated alkyl, halogenatedalkenyl, halogenated alkynyl, halogen, CN, NO₂, OR₁₁, SR₁₁, NR₁₁R₁₂,NH(CO)OR₁₁, NH(CO)NR₁₁R₁₂, NR₁₂(CO)R₁₁, O(CO)R₁₁, O(CO)OR₁₁, O(CS)R₁₁,NR₁₂(CS)R₁₁, NH(CS)NR₁₁R₁₂, NR₁₂(CS)OR₁₁. R₄ is preferably CN or NO₂. R₅is preferably trifluoromethyl, halogenated alkyl, halogenated alkenyl,halogenated alkynyl and halogen. R₆ and R₇ are preferably hydrogen,alkyl or halogen. R₄, R₅, R₆, and R₇ can be independently connected toform a cycle which can be aromatic, substituted aromatic, heterocyclicaromatic or non-aromatic, substituted heterocyclic aromatic ornon-aromatic, cycloalkyl, substituted cycloalkyl. X is selected fromsulfur (S), oxygen (O), NR wherein N is nitrogen and R₈ is selected fromthe group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, arylalkyl, arylalkenyl, arylalkynyl, halogenated alkyl,halogenated alkenyl, halogenated alkynyl, halogen, (CO)R₁₁, (CO)OR₁₁,(CS)R₁₁, (CS)OR₁₁.

R₁ is selected from hydrogen, aryl, substituted aryl, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,halogenated alkyl, halogenated alkenyl, halogenated alkynyl, arylalkyl,arylalkenyl, arylalkynyl, heterocyclic aromatic or non-aromatic,substituted heterocyclic aromatic or non-aromatic, cycloalkyl,substituted cycloalkyl, SO₂R₁₁, NR₁₁R₁₂, NR₁₂(CO)OR₁₁, NH(CO)NR₁₁R₁₂,NR₁₂(CO)R₁₁, O(CO)R₁₁, O(CO)OR₁₁, O(CS)R₁₁, NR₁₂(CS)R₁₁, NH(CS)NR₁₁R₁₂,NR₁₂(CS)OR₁₁. R₁ is preferably aryl, substituted aryl, alkyl,substituted alkyl, alkenyl, substituted alkenyl.

R₂ and R₃ are independently selected from hydrogen, aryl, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, halogenated alkyl, halogenated alkenyl, halogenated alkynyl,arylalkyl, arylalkenyl, arylalkynyl, heterocyclic aromatic ornon-aromatic, substituted heterocyclic aromatic or non-aromatic,cycloalkyl, substituted cycloalkyl. R₂ and R₃ can be connected to form acycle which can be heterocyclic aromatic or non aromatic, substitutedheterocyclic aromatic or non aromatic, cycloalkyl, substitutedcycloalkyl. R₁ and R₂ can be connected to form a cycle which can beheterocyclic aromatic or non aromatic, substituted heterocyclic aromaticor non aromatic.

A and B are independently selected from oxygen (O), sulfur (S) and N—R₉.R₉ is selected from hydrogen, aryl, substituted aryl, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,halogenated alkyl, halogenated alkenyl, halogenated alkynyl, arylalkyl,arylalkenyl, arylalkynyl, heterocyclic aromatic or non-aromatic,substituted heterocyclic aromatic or non-aromatic, cycloalkyl,substituted cycloalkyl, SO₂R₁₁, NR₁₁R₁₂, NR₁₂(CO)OR₁₁, NH(CO)NR₁₁R₁₂,NR₁₂(CO)R₁₁, O(CO)R₁₁, O(CO)OR₁₁, O(CS)R₁₁, NR₁₂(CS)R₁₁, NH(CS)NR₁₁R₁₂,NR₁₂(CS)OR₁₁.

R₁₁ and R₁₂, are independently selected from hydrogen, alkyl,substituted alkyl, alkenyl or substituted alkenyl, alkynyl orsubstituted alkynyl, aryl, substituted aryl, arylalkyl, arylalkenyl,arylalkynyl, heterocyclic aromatic or non-aromatic, substitutedheterocyclic aromatic or non-aromatic. R₁₁ and R₁₂ can be connected toform a cycle which can be heterocyclic aromatic or non-aromatic,substituted heterocyclic aromatic, cycloalkyl, substituted cycloalkyl.

The following definitions apply to the terms as used throughout thisspecification, unless otherwise limited in specific instances.

As used herein, the term “alkyl” denotes branched or unbranchedhydrocarbon chains, preferably having about 1 to about 8 carbons, suchas, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,tert-butyl, 2-methylpentyl, pentyl, hexyl, isohexyl, heptyl,4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl and the like.“Substituted alkyl” includes an alkyl group optionally substituted withone or more functional groups which are attached commonly to suchchains, such as, hydroxyl, bromo, fluoro, chloro, iodo, mercapto orthio, cyano, alkylthio, heterocyclyl, aryl, heteroaryl, carboxyl,carbalkoyl, alkyl, alkenyl, nitro, amino, alkoxyl, amido, and the liketo form alkyl groups such as trifluoromethyl, 3-hydroxyhexyl,2-carboxypropyl, 2-fluoroethyl, carboxymethyl, cyanobutyl and the like.

Unless otherwise indicated, the term “cycloalkyl” as employed hereinalone or as part of another group includes saturated or partiallyunsaturated (containing 1 or more double bonds) cyclic hydrocarbongroups containing 1 to 3 rings, including monocycloalkyl, bicycloalkyland tricycloalkyl, containing a total of 3 to 20 carbons forming therings, preferably 3 to 10 carbons, and which can be fused to 1 or 2aromatic rings as described for aryl, which include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyland cyclododecyl, cyclohexenyl. “Substituted cycloalkyl” includes acycloalkyl group optionally substituted with 1 or more substituents suchas halogen, alkyl, alkoxy, hydroxy, aryl, aryloxy, arylalkyl,cycloalkyl, alkylamido, alkanoylamino, oxo, acyl, arylcarbonylamino,amino, nitro, cyano, thiol and/or alkylthio and/or any of thesubstituents included in the definition of “substituted alkyl.” Forexample,

and the like.

Unless otherwise indicated, the term “alkenyl” as used herein by itselfor as part of another group refers to straight or branched chainradicals of 2 to 20 carbons, preferably 2 to 12 carbons, and morepreferably 2 to 8 carbons in the normal chain, which include one or moredouble bonds in the normal chain, such as vinyl, 2-propenyl, 3-butenyl,2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2-heptenyl,3-heptenyl, 4-heptenyl, 3-octenyl, 3-nonenyl, 4-decenyl, 3-undecenyl,4-dodecenyl, 4,8,12-tetradecatrienyl, and the like. “Substitutedalkenyl” includes an alkenyl group optionally substituted with one ormore substituents, such as the substituents included above in thedefinition of “substituted alkyl” and “substituted cycloalkyl.”

Unless otherwise indicated, the term “alkynyl” as used herein by itselfor as part of another group refers to straight or branched chainradicals of 2 to 20 carbons, preferably 2 to 12 carbons and morepreferably 2 to 8 carbons in the normal chain, which include one or moretriple bonds in the normal chain, such as 2-propynyl, 3-butynyl,2-butynyl, 4-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl,3-heptynyl, 4-heptynyl, 3-octynyl, 3-nonynyl, 4-decynyl, 3-undecynyl,4-dodecynyl and the like. “Substituted alkynyl” includes an alkynylgroup optionally substituted with one or more substituents, such as thesubstituents included above in the definition of “substituted alkyl” and“substituted cycloalkyl.”

The terms “arylalkyl”, “arylalkenyl” and “arylalkynyl” as used alone oras part of another group refer to alkyl, alkenyl and alkynyl groups asdescribed above having an aryl substituent. Representative examples ofarylalkyl include, but are not limited to, benzyl, 1- and 2-phenylethyl,2- and 3-phenylpropyl, benzhydryl and naphthylmethyl and the like.“Substituted arylalkyl” includes arylalkyl groups wherein the arylportion is optionally substituted with one or more substituents, such asthe substituents included above in the definition of “substituted alkyl”and “substituted cycloalkyl.”

The term “halogen” or “halo” as used herein alone or as part of anothergroup refers to chlorine, bromine, fluorine, and iodine.

The terms “halogenated alkyl”, “halogenated alkenyl” and “halogenatedalkynyl” as used herein alone or as part of another group refers to“alkyl”, “alkenyl” and “alkynyl” which are substituted by one or moreatoms selected from fluorine, chlorine, bromine, and iodine.

Unless otherwise indicated, the term “aryl” or “Ar” as employed hereinalone or as part of another group refers to monocyclic and polycyclicaromatic groups containing 6 to 10 carbons in the ring portion (such asphenyl or naphthyl including 1-naphthyl and 2-naphthyl) and canoptionally include one to three additional rings fused to a carbocyclicring or a heterocyclic ring (such as aryl, cycloalkyl, heteroaryl orcycloheteroalkyl rings).

“Substituted aryl” includes an aryl group optionally substituted withone or more functional groups, such as halo, alkyl, haloalkyl, alkoxy,haloalkoxy, alkenyl, trifluoromethyl, trifluoromethoxy, alkynyl,cycloalkyl, cycloalkylalkyl, cycloheteroalkyl, cycloheteroalkylalkyl,aryl, heteroaryl, arylalkyl, aryloxy, aryloxyalkyl, arylalkoxy,alkoxycarbonyl, arylcarbonyl, arylalkenyl, aminocarbonylaryl, arylthio,arylsulfinyl, arylazo, heteroarylalkyl, heteroarylalkenyl,heteroarylheteroaryl, heteroaryloxy, hydroxy, nitro, cyano, amino,substituted amino wherein the amino includes 1 or 2 substituents (whichare alkyl, aryl or any of the other aryl compounds mentioned in thedefinitions), carbamoyl, alkyl carbamoyl, amidified carboxy, amidifiedcarboxyalkyl, alkyl amidified carboxyalkyl, thiol, alkylthio, arylthio,heteroarylthio, arylthioalkyl, alkoxyarylthio, alkylcarbonyl,arylcarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl,aminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy, alkylcarbonylamino,arylcarbonylamino, arylsulfinyl, arylsulfinylalkyl, arylsulfonylamino orarylsulfonaminocarbonyl and/or any of the alkyl substituents set outherein.

Unless otherwise indicated, the term “heterocyclic” or “heterocycle”, asused herein, represents an unsubstituted or substituted stable 5- to10-membered monocyclic ring system which can be saturated orunsaturated, and which consists of carbon atoms and from one to fourheteroatoms selected from N, O, or S, and wherein the nitrogen andsulfur heteroatoms can optionally be oxidized; and the nitrogenheteroatom can optionally be quaternized. The heterocyclic ring can beattached at any heteroatom or carbon atom which results in the creationof a stable structure. Examples of such heterocyclic groups include, butare not limited to, piperidinyl, piperazinyl, oxopiperazinyl,oxopiperidinyl, oxopyrrolidinyl, azepinyl, oxoazepinyl, pyrrolyl,pyrrolidinyl, furanyl, thienyl, pyrazolyl, pyrazolidinyl, imidazolyl,imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl,pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl,morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, thiadiazolyl,tetrahydropyranyl, thiamorpholinyl, thiamorpholinyl sulfoxide,thiamorpholinyl sulfone, and oxadiazolyl. The term “heterocyclicaromatic” as used here in alone or as part of another group refers to a5- or 7-membered aromatic ring which includes 1, 2, 3 or 4 hetero atomssuch as nitrogen, oxygen or sulfur and such rings fused to an aryl,cycloalkyl, heteroaryl or heterocycloalkyl ring (e.g., benzothiophenyl,indolyl), and includes possible N-oxides. “Substituted heteroaryl”includes a heteroaryl group optionally substituted with 1 to 4substituents, such as the substituents included above in the definitionof “substituted alkyl” and “substituted cycloalkyl.” Examples ofheteroaryl groups include the following:

and the like.

The compounds of formula II can be present as salts, which are alsowithin the scope of this invention. Pharmaceutically acceptable (i.e.,non-toxic, physiologically acceptable) salts are preferred. If thecompounds of formula II have, for example, at least one basic center,they can form acid addition salts. These are formed, for example, withstrong inorganic acids, such as mineral acids, for example sulfuricacid, phosphoric acid or a hydrohalic acid, with strong organiccarboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atomswhich are unsubstituted or substituted, for example, by halogen, forexample acetic acid, such as saturated or unsaturated dicarboxylicacids, for example oxalic, malonic, succinic, maleic, fumaric, phthalicor terephthalic acid, such as hydroxycarboxylic acids, for exampleascorbic, glycolic, lactic, malic, tartaric or citric acid, such asamino acids, (for example aspartic or glutamic acid or lysine orarginine), or benzoic acid, or with organic sulfonic acids, such as(C1-C4) alkyl or arylsulfonic acids which are unsubstituted orsubstituted, for example by halogen, for example methyl- orp-toluene-sulfonic acid. Corresponding acid addition salts can also beformed having, if desired, an additionally present basic center. Thecompounds of formula II having at least one acid group (for exampleCOOH) can also form salts with bases. Suitable salts with bases are, forexample, metal salts, such as alkali metal or alkaline earth metalsalts, for example sodium, potassium or magnesium salts, or salts withammonia or an organic amine, such as morpholine, thiomorpholine,piperidine, pyrrolidine, a mono, di or tri-lower alkylamine, for exampleethyl, tert-butyl, diethyl, diisopropyl, triethyl, tributyl ordimethyl-propylamine, or a mono, di or trihydroxy lower alkylamine, forexample mono, di or triethanolamine. Corresponding internal salts canfurthermore be formed. Salts which are unsuitable for pharmaceuticaluses but which can be employed, for example, for the isolation orpurification of free compounds of formula II or their pharmaceuticallyacceptable salts, are also included. Preferred salts of the compounds offormula II which contain a basic group include monohydrochloride,hydrogensulfate, methanesulfonate, phosphate or nitrate. Preferred saltsof the compounds of formula II which contain an acid group includesodium, potassium and magnesium salts and pharmaceutically acceptableorganic amines.

The term “modulator” used in this invention refers to a chemicalcompound with capacity to either enhance (e.g., “agonist” activity) orinhibit (e.g., “antagonist” activity) a functional property ofbiological activity or process (e.g., enzyme activity or receptorbinding); such enhancement or inhibition can be contingent on theoccurrence of a specific event, such as activation of a signaltransduction pathway, and/or can be manifest only in particular celltypes.

The term “prodrug esters” as employed herein includes imines, esters andcarbonates formed by reacting one or more hydroxyls of compounds offormula II with alkyl, alkoxy, or aryl substituted acylating agentsemploying procedures known to those skilled in the art to generateacetates, pivalates, methylcarbonates, benzoates and the like. Anycompound that can be converted in vivo to provide the bioactive agent(i.e., the compound of formula II) is a prodrug within the scope andspirit of the invention. Various forms of prodrugs are well known in theart. A comprehensive description of prodrugs and prodrug derivatives aredescribed in: (1) The Practice of Medicinal Chemistry, Camille G.Wermuth et al., Ch 31, (Academic Press, 1996); (2) Design of Prodrugs,edited by H. Bundgaard, (Elsevier, 1985); (3) A Textbook of Drug Designand Development, P. Krogsgaard-Larson and H. Bundgaard, eds. Ch 5, pgs113-191 (Harwood Academic Publishers, 1991).

SYNTHESIS

The compounds of formula II of the invention can be prepared as shown inthe following reaction schemes and description thereof, as well asrelevant published literature procedures that can be used by one skilledin the art. Exemplary reagents and procedures for these reactions appearhereinafter and in the working Examples.

As illustrated in Scheme 1, compounds of formula A4 can be prepared fromintermediate A3 with an appropriate electrophile. Intermediates offormula A3 can be obtained by reacting intermediates A1 with A2 in anappropriate solvent such as N,N-dimethylformamide. Intermediates A1 andA2 can be obtained commercially, can be prepared by methods known in theliterature, or can be readily prepared by one skilled in the art.Compounds of formula A3 can be treated with acid to afford compounds offormula A5. Compounds of formula A5 can be treated with Lawesson'sreagent to obtain compounds of formula A6.

Synthesis of 3-(trifluoromethyl)pyridine-N-oxide, A8

To a mixture of 3-(trifluoromethyl)pyridine A7 (1.47 g, 10 mmol) andmethyltrioxorhenium (0.0025 g, 0.01 mmol) in dichloromethane (2 ml) wasadded 30% hydrogen peroxide (4 ml). The mixture was stirred at roomtemperature for 5 hours. A small portion of MnO₂ (3 mg) was added andthe medium was stirred for an additional 1 hour and then dichloromethanewas added (50 ml). The medium was washed with brine, dried over MgSO₄and concentrated to obtain compound A8 as an off-white powder (1.56 g,9.6 mmol, 96%). ¹H NMR (400 MHz, CDCl₃) δ 7.22-7.23 (m, 2H), 8.15 (d,J=3.6, 1H), 8.23 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 120.50 (q, J=3.5Hz), 121.58 (q, J=271.4 Hz), 126.48, 130.10 (q, J=34.5 Hz), 136.52 (q,=3.7 Hz), 141.89.

Synthesis of 2-cyano-3-(trifluoromethyl)pyridine, A9

To a solution of 3-(trifluoromethyl)pyridine-N-oxide A8 (1.3 g, 8 mmol)in acetonitrile was added trimethylsilyl cyanide (0.99 g, 10 mmol) andtriethylamine (2.02 g, 20 mmol). The mixture was stirred at roomtemperature for 24 hours and then was washed with saturated Na₂CO₃ andextracted with dichloromethane. The organic layer was dried over MgSO₄and concentrated to yield a brown residue which was chromatographed(EtOAc:Pentane, 1:2). Compound A9 was obtained as a light yellow solid(0.715 g, 4.16 mmol, 52%). ¹H NMR (400 MHz, CDCl₃) δ 7.73 (dd, J₁=8.0Hz, J₂=4.8 Hz, 1H), 8.15 (d, J=8.0 Hz, 1H), 8.91 (d, J=4.8 Hz, 1H); ¹³CNMR (100 MHz, CDCl₃) δ 114.18, 121.74 (q, J=272.3 Hz), 126.65, 130.45(q, J=33.8 Hz), 131.25, 134.66 (q, J=4.2 Hz), 153.44.

Synthesis of 2-cyano-3-(trifluoromethyl)-5-nitropyridine, A10

To a mixture of A9 (0.688 g, 4 mmol) and tetramethylammonium nitrate(1.09 g, 8 mmol) in 1,2-dichloroethane was added trifluoroaceticanhydride (1.68 g, 8 mmol). The mixture was sealed and heated to 60° C.for 48 hours. The mixture was washed with saturated sodium bicarbonateand extracted with ethyl acetate. The organic layer was dried over MgSO₄and concentrated to yield a yellow residue which was chromatographed(EtOAc:pentane, 1:4) to yield compound A10 (0.095 g, 0.44 mmol, 11%) andthe remaining starting material. ¹H NMR (400 MHz, CDCl₃) δ 8.91 (d,J=2.4 Hz, 1H), 9.69 (d, J=2.4 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ112.70, 120.65 (q, J=273.5 Hz), 129.11, 130.40 (q, J=4.4 Hz), 131.58 (q,J=35.5 Hz), 144.22, 148.23.

Synthesis of 2-cyano-3-(trifluoromethyl)-5-aminopyridine, A11

A mixture of 2-cyano-3-(trifluoromethyl)-5-nitropyridine A10 (0.095 g,0.44 mmol) and iron powder (0.112 g, 2 mmol) in ethyl acetate (1 ml) andacetic acid (1 ml) was heated for 15 hours. The solid particle wasfiltered through Celite and the filtrate was concentrated andchromatographed (EtOAc:pentane, 1:1) to yield compound A11 (0.075 g, 0.4mmol, 91%). ¹H NMR (400 MHz, CDCl₃) δ 6.36 (bs, 2H), 7.38 (d, J=2.4 Hz,1H), 8.26 (d, J=2.4 Hz, 1H).

Alternatively, 2-cyano-3-(trifluoromethyl)-5-nitropyridine A10 can bereacted with hydrogen over Raney-Ni to obtain2-cyano-3-(trifluoromethyl)-5-aminopyridine, A11.

Synthesis of 5-isothiocyanato-3-trifluoromethylpyridine-2-carbonitrile,A12

To a heterogeneous mixture of2-cyano-3-(trifluoromethyl)-5-nitropyridine A11 (0.075 g, 0.4 mmol) inwater (2 ml) was added thiophosgene (50 μl). The mixture was stirred for2 hours and then washed with water and extracted with chloroform. Theorganic layer was dried over MgSO₄ and concentrated to yield compoundA12 (0.087 g, 0.38 mmol, 95%). ¹H NMR (400 MHz, CDCl₃) δ 7.85 (d, J=2.4Hz, 1H), 8.72 (d, J=2.4 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 113.61,121.04 (q, J=273.1 Hz), 127.41, 130.38 (q, J=4.3 Hz), 131.44 (q, J=34.4Hz), 133.55, 144.75, 150.30.

Synthesis of 1-(4-methylphenyl)aminocyclobutanenitrile, B1

Trimethylsilyl cyanide (0.93 ml, 7 mmol) was added dropwise to a mixtureof p-toluidine (0.535 g, 5 mmol) and cyclobutanone (0.42 g, 6 mmol). Thereaction mixture was stirred at room temperature for 6 h and thenconcentrated under vacuum to obtain a brown liquid which was subjectedto chromatography (dichloromethane) to yield B1 (0.912 g, 4.9 mmol, 98%)as a yellowish solid.

Synthesis of5-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7yl)-3-trifluoromethylpyridine-2-carbonitrile,A51

A mixture of A12 (0.057 g, 0.265 mmol) and B1 (0.05 g, 0.265 mmol) inDMF (0.5 ml) was stirred at room temperature for 24 h. To this mixturewere added methanol (2 ml) and aq. 2N HCl (1 ml). The second mixture wasrefluxed for 2 h. After being cooled to room temperature, the reactionmixture was poured into cold water (10 ml) and extracted with ethylacetate (20 ml). The organic layer was dried over MgSO₄, concentratedand chromatographed (dichloromethane) to yield compound A51 (0.066 g,0.159 mmol, 60%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.63-1.73 (m, 1H), 2.17-2.28 (m, 1H), 2.47 (s,3H), 2.55-2.71 (m, 4H), 7.21 (d, J=8.4 Hz, 2H), 7.41 (d, J=8.4 Hz, 2H),8.39 (d, J=2.0 Hz, 1H), 9.11 (d, J=2.0 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz)δ 13.70, 21.38, 31.46, 67.61, 113.88, 121.36 (q, J=272.9 Hz), 129.45,129.73, 130.40 (q, J=34.3 Hz), 130.86, 132.14, 132.53, 134.04 (q, J=4.3Hz), 140.33, 152.37, 174.74, 179.17.

N-methyl-4-(1-cyanocyclobutylamino)-2-fluorobenzamide, B2

Sodium cyanide (1.47 g, 30 mmol) was added to a mixture of N-methyl4-amino-2-fluorobenzamide (1.68 g, 10 mmol) and cyclobutanone (1.4 g, 20mmol) in 90% acetic acid (20 ml). The reaction mixture was stirred at80° C. for 24 hours. The mixture was washed with water and extractedwith ethyl acetate. The organic layer was dried over magnesium sulfateand concentrated to dryness under vacuum. The solid was washed with a50:50 mixture of ethyl ether and hexane (10 ml) to remove cyclobutanonecyanohydrin to afford after filtration B2 (2.19 g, 8.87 mmol, 89%). ¹HNMR (CDCl₃, 400 MHz) δ 1.87-1.95 (m, 1H), 2.16-2.27 (m, 1H), 2.35-2.41(m, 2H), 2.76-2.83 (m, 2H), 2.97 (d, J=4.4 Hz, 3H), 4.68 (bs, 1H), 6.29(dd, J=14.3, 1.8 Hz, 1H), 6.48 (dd, J=8.3, 1.8 Hz, 1H), 6.75 (q, J=4.4Hz, 1H), 7.90 (dd, J=8.3, 8.3 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 15.7,26.7, 33.9, 49.4, 100.2 (d, J=29.5 Hz), 110.6, 111.0 (d, J=11.8 Hz),133.1 (d, J=4.2 Hz), 148.4 (d, J=12.0 Hz), 162.0 (d, J=244.1 Hz), 164.4(d, J=3.6 Hz).

Synthesis of4-[7-(6-cyano-5-trifluoromethylpyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-2-fluoro-N-methylbenzamide,A52

A mixture of A12 (0.03 g, 0.13 mmol) and B2 (0.032 g, 0.13 mmol) in DMF(0.5 ml) was heated under microwave irradiation at 80° C. for 20 hours.To this mixture was added methanol (2 ml) and aq. 2N HCl (1 ml). Thesecond mixture was refluxed for 2 hours. After being cooled to roomtemperature, the reaction mixture was poured into cold water (10 ml) andextracted with ethyl acetate (15 ml). The organic layer was dried overMgSO₄, concentrated and chromatographed (dichloromethane:acetone, 95:5)to yield A52 (0.022 g, 0.046 mmol, 35%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.66-1.76 (m, 1H), 2.19-2.31 (m, 1H),2.51-2.60 (m, 2H), 2.67-2.75 (m, 2H), 3.07 (d, J=4.9 Hz, 3H), 6.75 (q,J=4.8 Hz, 1H), 7.17 (dd, J=11.4, 1.9 Hz, 1H), 7.26 (dd, J=8.3, 1.9 Hz,1H), 8.31 (dd, J=8.3, 8.3 Hz, 1H), 8.34 (d, J=2.1 Hz, 1H), 9.08 (d,J=2.1 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.6, 27.0, 31.7, 67.6, 113.7,118.1, 118.4, 121.4 (q, J=272.9 Hz), 126.5, 130.0, 130.5 (q, J=34.5 Hz),132.2, 133.7, 134.0, (q, J=4.2 Hz), 138.7 (d, J=10.7 Hz), 152.2, 160.5(d, J=249.4 Hz), 162.6, 174.1, 179.0; ¹⁹F NMR (CDCl₃, 100 MHz) δ−110.94, −62.57.

Scheme 3 Synthesis of A52

In other embodiments, the present invention is directed to the method ofsynthesizing A52 described below. In some embodiments, Examples 1-8 canbe performed sequentially to synthesize A52. However, as one of skill inthe art will appreciate, this invention is not limited to the steps inExamples 1-8 as equivalent steps to those below are also encompassed bythe present invention. Persons skilled in the art will recognizeadditional compounds that can be prepared utilizing similar methodology.

Synthesis of 3-(trifluoromethyl)pyridin-2(1H)-one, 2

A solution of 2-chloro-3-(trifluoromethyl)pyridine 1 (5.00 g, 27.54mmol) in a mixture of glacial acetic acid (50 ml) and water (5 ml) wasrefluxed for 7 days. The mixture was diluted with water (100 ml) and 6Naqueous NaOH was added until a pH of about 5 to about 6 was reached. Themixture was extracted with ethyl acetate (3×40 ml), the combined organicphases were dried over Na₂SO₄, and then all solvents were removed underreduced pressure. The resulting residue was dissolved in ethyl acetateand hexane was added to precipitate a product. After filtration,3-(trifluoromethyl)pyridin-2(1H)-one 2 was obtained as an off-whitepowder (4.16 g, 25.51 mmol, 93%).

¹H NMR (400 MHz, DMSO) δ 12.31 (bs, 1H), 7.91 (d, J=7.1 Hz, 1H), 7.69(d, J=6.4 Hz, 1H), 6.30 (t, J=6.7 Hz, 1H).¹

Synthesis of 5-nitro-3-(trifluoromethyl)pyridin-2(1H)-one, 3

A mixture of 3-(trifluoromethyl)pyridin-2(1H)-one 2 (2.00 g, 12.26 mmol)and sulfuric acid (H₂SO₄, 3.5 ml, 30%) was heated to 90° C. and nitricacid (HNO₃, 2.5 ml, 65%) was added. The mixture was stirred at 90° C.for 8 hours and additional nitric acid (I ml, 65%) was added. Themixture was stirred for an additional 6 hours at 90° C. and was thenpoured into a beaker containing ice (30 ml). The mixture was dilutedwith water (30 ml) and 6N aqueous NaOH was added until a pH of about 4to about 5. The mixture was extracted with ethyl acetate (3×40 ml), thecombined organic phases dried over Na₂SO₄ and all solvents were removedunder reduced pressure. The residue was dissolved in ethyl acetate andthe product precipitated by the addition of hexane. After filtration,5-nitro-3-(trifluoromethyl)pyridin-2(1H)-one 3 was obtained as a yellowpowder (1.58 g, 7.59 mmol, 62%).

¹H NMR (400 MHz, DMSO) δ 13.47 (bs, 1H), 8.95 (d, J=2.7 Hz, 1H), 8.46(d, J=2.5 Hz, 1H).²

Synthesis of 2-chloro-5-nitro-3-(trifluoromethyl)pyridine, 4

A mixture of 5-nitro-3-(trifluoromethyl)pyridin-2(1H)-one 3 (1.50 g,7.21 mmol), POCl₃ (2.76 g, 18.02 mmol) and PCl₅ (1.4 g, 10.09 mmol) isheated to about 110-120° C. for 8 hours and then poured into ice water.The mixture is neutralized with solid NaHCO₃ and extracted with ethylacetate (3×40 ml). The combined organic phases is dried over Na₂SO₄ andall solvents removed under reduced pressure to obtain2-chloro-5-nitro-3-(trifluoromethyl)pyridine 4.

Synthesis of 6-chloro-5-(trifluoromethyl)pyridin-3-amine, 5

2-Chloro-5-nitro-3-(trifluoromethyl)pyridine 4 (1.57 g, 6.93 mmol) isdissolved in tetrahydrofuran (THF) (10 ml) and added to a suspension ofRaney-Ni (200 mg) in THF (20 ml). Hydrogen gas is slowly bubbled throughthe stirred solution for 24 hours using a balloon. The mixture isfiltered through Celite® (available from World Minerals, Inc., Lompoc,Calif.) and the solvent is removed under reduced pressure to obtain6-chloro-5-(trifluoromethyl)pyridin-3-amine 5.

Synthesis of 1,1-dimethylethylcarbamateN-6-chloro-5-(trifluoromethyl)pyridin-3-yl, 6

The crude 6-chloro-5-(trifluoromethyl)pyridin-3-amine 5 (1.3 g crude,6.61 mmol) is dissolved in pyridine (10 ml) and 4-dimethylaminopyridine(DMAP) (50 mg) is added. Di-tert-butyl dicarbonate (2.17 g) is addeddropwise and mixture stirred at 22° C. for 4 hours. Toluene (20 ml) isadded and all solvents is removed under reduced pressure. The residue isfiltered through a plug of silica gel (hexane/ethyl acetate 2:1) toobtain tert-butyl N-6-chloro-5-(trifluoromethyl)pyridin-3-ylcarbamate 6.

Synthesis of 5-amino-3-(trifluoromethyl)pyridine-2-carbonitrile, 8

The crude tert-butyl N-6-chloro-5-(trifluoromethyl)pyridin-3-ylcarbamate6 (2.4 g, 6.61 mmol) is dissolved in dimethylacetamide (DMA) (25 ml) andphenanthroline (120 mg, 0.66 mmol) is added. The mixture is heated to80° C. and KCN (0.47 g, 7.27 mmol) is added. After stirring the mixturestirred for 10 min, CuCN (118 mg, 0.13 mmol) is added and the mixturestirred for 2 hours at 110° C. The cooled mixture is poured into aphosphate buffer (150 ml, pH 7), ethyl acetate (50 ml) is added and themixture is filtered through Celite®. The layers are separated and theaqueous phase is extracted with ethyl acetate (3×40 ml). The combinedorganic phases are washed with saturated aqueous NaCl (4×30 ml), driedover Na₂SO₄ and all solvents removed under reduced pressure to producethe crude N-t-butoxycarbonyl nitrile 7.

The crude N-t-butoxycarbonyl nitrile 7 is dissolved in dichloromethane(20 ml) and trifluoroacetic acid (TFA) (4 ml is added. The mixture isstirred for 3 hours and evaporated. The residue is purified by columnchromatography on silica gel (hexane/ethyl acetate 2:1) to obtain5-amino-3-(trifluoromethyl)pyridine-2-carbonitrile 8.

Synthesis of5-isothiocyanato-3-(trifluoromethyl)pyridine-2-carbonitrile, 9

5-Amino-3-(trifluoromethyl)pyridine-2-carbonitrile 8 (1.141 g, 6.1 mmol)is mixed with chloroform (5 ml) and water (40 ml) to give a whitesuspension. Thiophosgene (0.701 ml, 9.15 mmol) is added and the reactionstirred for 2 hours at 22° C. to give a clear biphasic system.Chloroform (20 ml) is added and the phases are separated. The aqueouslayer is extracted with chloroform (30 ml) and the combined organic iswashed with saturated aqueous NaHCO₃ and water, dried over MgSO₄ and thesolvent is removed under reduced pressure. The crude5-isothiocyanato-3-(trifluoromethyl) pyridine-2-carbonitrile 9 is driedunder vacuum and used as such in the next step, for example, in the stepdescribed in Example 8 below.

Synthesis of4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzamide11, A52

Crude 5-isothiocyanato-3-(trifluoromethyl)picolinonitrile 9 (1.390 g,6.07 mmol) is placed in a 50 mL round-bottomed flask and4-(1-cyanocyclobutylamino)-2-fluoro-N-methylbenzamide 10 (0.5 g, 2.022mmol) is added to the flask. The mixture is left under vacuum (using anoil pump) for 1 hour. N,N-dimethylformamide (DMF) (6 ml) is added, theflask sealed under argon with a stopper and heated to 80° C. in a CEMmicrowave reactor for 20 hours. Methanol (10 ml) and 2N HCl (6 ml) isadded and the mixture is refluxed for 2 hours. The mixture is dilutedwith water (30 ml) and saturated aqueous NaHCO₃ (30 ml) is added. Themixture is extracted with ethyl acetate (3×20 ml).

The combined organic layers is washed with saturated aqueous NaCl (20ml), dried over. Na₂SO₄, filtered and concentrated under reducedpressure. The crude product is purified by column chromatography onsilica gel (dichloromethane/acetone 95:5) to obtain4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzamide11.

Scheme 4 Synthesis of A52 Example 1 Synthesis of2-bromo-5-nitro-3-(trifluoromethyl)pyridine, 21

5-nitro-3-(trifluoromethyl)pyridin-2(1H)-one 3 is obtained by the routesprovided in Examples 1 and 2 of Scheme 3, above.

A mixture of 5-nitro-3-(trifluoromethyl)pyridin-2(1H)-one 3, POBr₃ (1.5equivalents), PBr₃ (4 equivalents), and Br₂ (2 equivalents) is heated toabout 90-110° C. and is then poured into ice water. The mixture isneutralized and extracted. The combined organic phases are dried overNa₂SO₄ and all solvents removed under reduced pressure to obtain2-bromo-5-nitro-3-(trifluoromethyl)pyridine 21 in a yield of 88%.

Alternatively, POBr₃ is substituted by POCl₃ to yield a mixture in theproduct having a ratio of bromine to chlorine substituents of 6:1 orbetter.

Synthesis of 5-nitro-3-(trifluoromethyl)pyridine-2-carbonitrile, 22

The crude 2-bromo-5-nitro-3-(trifluoromethyl)pyridine 21 is dissolved indimethylacetamide (DMA) and phenanthroline (0.2 equivalents) is added.The mixture is heated to 160° C. and CuCN (2 equivalents) is added. Themixture is stirred for 40 minutes. Chromatography is performed toproduce the 5-nitro-3-(trifluoromethyl)pyridine-2-carbonitrile 22 in ayield of 67%.

Synthesis of 5-amino-3-(trifluoromethyl)pyridine-2-carbonitrile, 8

A mixture of 5-nitro-3-(trifluoromethyl)pyridine-2-carbonitrile 22 andiron powder in acetic acid is heated.5-amino-3-(trifluoromethyl)pyridine-2-carbonitrile, 8 is obtained in ayield of 91%.

Synthesis of4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzamide11, A52

5-amino-3-(trifluoromethyl)pyridine-2-carbonitrile 8 is treated asdiscussed in Example 7 of Scheme 3, above, to obtain5-isothiocyanato-3-(trifluoromethyl)pyridine-2-carbonitrile 9.

5-isothiocyanato-3-(trifluoromethyl)pyridine-2-carbonitrile, 9 isreacted with 4-(1-cyanocyclobutylamino)-2-fluoro-N-methylbenzamide 10 asdiscussed in Example 8 of Scheme 3, above, to obtain4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzamide11 (A52).

Scheme 5 Alternative Synthesis of A52 Synthesis of3-(trifluoromethyl)-5-isothiocyanatopyridine-2-carbonitrile (A)

A solution of 2-hydroxy-3-(trifluoromethyl)pyridine C in a mixture ofN-iodosuccinimide (NIS), acetonitrile, and dimethylformamide (DMF) isheated at 80° C. for 2 hours to produce2-hydroxy-3-trifluoromethyl-5-(iodo)pyridine I (greater than 80% yield).The 2-hydroxy-3-trifluoromethyl-5-(iodo)pyridine I is then mixed withPOCl₃ in DMF and heated to 130° C. in a microwave for 20 minutes toproduce 2-chloro-3-trifluoromethyl-5-(iodo)pyridine J (yield of 50 to55%). The 2-chloro-3-trifluoromethyl-5-(iodo)pyridine K is reacted in asolution of pMBnNH₂, palladium(II) acetate,2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP), triethylamine, andcesium carbonate in toluene to produce5-((4-methoxyphenyl))methylamino)-2-chloro-3-(trifluoromethyl)pyridine K(yield of 40%). The5-((4-methoxyphenyl))methylamino)-2-chloro-3-(trifluoromethyl)pyridine Kis reacted in a solution of zinc cyanide,tris(dibenzylideneacetone)dipalladium (Pd₂(dba)₃), and 1,1′-bis(diphenylphosphino)ferrocene (dppf) in DMF to provide5-(4-methoxybenzylamine)-2-cyano-3-(trifluoromethyl)pyridine K (yield of92%). The 5-(4-methoxybenzylamine)-2-cyano-3-(trifluoromethyl)pyridine Kis reacted in a solution of dichloromethane and trifluoroacetic acid toprovide 2-cyano-3-trifluoromethyl-5-(amino)pyridine H (yield greaterthan 95%). The 2-cyano-3-trifluoromethyl-5-(amino)pyridine H is reactedwith thiophosgene in water at 25° C. for 2 hours to provide5-isothiocyanato-3-(trifluoromethyl)pyridine-2-carbonitrile A (yield of74% to 95%).

Synthesis of 4-(1-cyanocyclobutylamino)-2-fluoro-N-methylbenzamideintermediate B

A solution of 2,4-difluoro-benzoylchloride D in a solution ofmethylamine and tetrahydrofuran (THF) is allowed to react to produce2,4-difluoro-N-methylbenzamide M (quantitative yield). The2,4-difluoro-N-methylbenzamide M is mixed with in a solution ofacetonitrile and 4-methoxy-benzenemethanamine and heated in a microwavefor 20 minutes at 190° C. to produce2-fluoro-4-(4-methoxybenzylamino)-N-methylbenzamide S (yield of 40%).The 2-fluoro-4-(4-methoxybenzylamino)-N-methylbenzamide S is reacted ina solution of dichloromethane and trifluoroacetic acid to produce2-fluoro-4-amino-N-methylbenzamide T (yield greater than 95%). The2-fluoro-4-amino-N-methylbenzamide T is reacted with a solution ofsodium cyanide and cyclobutanone to produce4-(1-cyanocyclobutylamino)-2-fluoro-N-methylbenzamide B.

Coupling of A and B to produce4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzamide,A52

5-isothiocyanato-3-(trifluoromethyl)pyridine-2-carbonitrile, 9, A isreacted with 4-(1-cyanocyclobutylamino)-2-fluoro-N-methylbenzamide B inDMF solution by heating in a microwave at 80° C. for 20 hours. Methanoland hydrochloric acid are then added and the reaction allowed to proceedfor 2 hours to produce4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzamide,A52 (yield 35 to 87%).

ACTIVITY Utility

The compounds of the present invention modulate the function of thenuclear hormone receptors, particularly the androgen receptor, andinclude compounds which are, for example, selective agonists orselective antagonists of the androgen receptor (AR). Thus, the presentcompounds are useful in the treatment of AR-associated conditions. An“AR-associated condition,” as used herein, denotes a condition ordisorder which can be treated by modulating the function or activity ofan AR in a subject, wherein treatment comprises prevention, partialalleviation or cure of the condition or disorder. Modulation can occurlocally, for example, within certain tissues of the subject, or moreextensively throughout a subject being treated for such a condition ordisorder. Preferably, the compounds with potent antagonistic activityare used for the treatment of androgen related prostate cancer.

Combination

The present invention includes within its scope pharmaceuticalcompositions comprising, as an active ingredient, a therapeuticallyeffective amount of at least one of the compounds of formula II, aloneor in combination with a pharmaceutical carrier or diluent. Optionally,compounds of the present invention can be used alone, in combinationwith other compounds of the invention, or in combination with one ormore other therapeutic agent(s), e.g., an antibiotic or otherpharmaceutically active material.

Pharmacological Assay

The compounds in this invention were identified through screening onhormone sensitive and hormone refractory prostate cancer cells forantagonistic and agonistic activities. The compounds with antagonistactivity are potential drugs for the treatment of prostate cancer, bothhormone sensitive and hormone refractory.

The biological activity of the compound of formula II was measured bysecreted levels of prostate specific antigen (PSA). It is wellestablished that PSA levels are indicators of AR activities in prostatecancer. To examine if the compounds affect AR function in aphysiological environment, we determined secreted levels of endogenousPSA induced by R1881 in the hormone sensitive (HS) and hormonerefractory (HR) cancer cells. HR cells are LNCaP cells engineered toexpress elevated levels of androgen receptor protein (LNCaP/AR cells),analogous to levels observed in patients with HR cancer who relapsewhile taking current antiandrogens such as bicalutamide, which acquireagonist properties when AR is highly expressed. LNCaP cells (or LNCaP/ARcells) were maintained in Iscove's medium containing 10% FBS. Five daysprior to drug treatment, the cells were grown in Iscove's mediumcontaining 10% CS-FBS to deprive of androgens. The cells were split andgrown in Iscove's medium containing 10% CS-FBS with appropriateconcentrations of R1881 and the test compounds. After 5 days ofincubation, secreted PSA levels were assayed using PSA ELISA kits(American Qualex, San Clemente, Calif.) (See FIG. 1 and FIG. 3). The MTSassay was also used to examine the growth inhibition of the compounds offormula II (See FIG. 2).

Pharmacokinetic Data

The pharmacokinetics of A52 was evaluated in vivo using 8 week-old FVBmice which were purchased from Charles River Laboratories. Mice weredivided into groups of three for each time point (See FIG. 4). Two micewere not treated with drug and two other mice were treated with vehiclesolution. Each group was treated with 10 mg per kilogram of body weight.The drug was dissolved in a mixture 50:10:1:989 ofDMSO:Carboxymethylcellulose:T Tween 80:H₂0 (Vehicle solution) and wasadministered orally. After drug administration, the animals wereeuthanized via CO₂ inhalation at different timepoints: 1 min, 5 min, 15min, 30 min, 2 h, 4 h, 8 h, 16 h. Animals were immediately bleed afterexposure to CO₂ via cardiac puncture (1 ml BD syringe+27G 5/8 needle).

The serum samples were analyzed to determine the drug's concentration bythe HPLC which (Waters 600 pump, Waters 600 controller and Waters 2487detector) was equipped with an Alltima C18 column (3μ, 150 mm×4.6 mm).All RD compounds were detected at 254 nm wave length and bicalutamidewas detected at 270 nm wave length.

The samples for HPLC analysis were prepared according to the followingprocedure:

Blood cells were separated from serum by centrifugation.

To 400 μl of serum were added 80 μl of a 10 μM solution of RD75 inacetonitrile as internal standard and 520 μl of acetonitrile.Precipitation occurred.

The mixture was vortexed for 3 minutes and then placed under ultrasoundfor 30 minutes.

The solid particles were filtered off or were separated bycentrifugation.

The filtrate was dried under an argon flow to dryness. The sample wasreconstructed to 80 μl with acetonitrile before analyzing by HPLC todetermine the drug concentration.

Standard curve of drug was used to improve accuracy.

In Vivo Assay

All animal experiments were performed in compliance with the guidelinesof the Animal Research Committee of the University of California at LosAngeles. Animals were bought from Taconic and maintained in a laminarflow tower in a defined flora colony. LNCaP-AR and LNCaP-vector cellswere maintained in RPMI medium supplemented with 10% FBS. 10⁶ cells in100 μl of 1:1 Matrigel to RPMI medium were injected subcutaneously intothe flanks of intact or castrated male SCID mice. Tumor size wasmeasured weekly in three dimensions (length×width×depth) using calipers.Mice were randomized to treatment groups when tumor size reachedapproximately 100 mm³. Drugs were given orally everyday at the dose of10 mg/kg. (See FIG. 5 and FIG. 6) At a daily dose of 10 mg/kg, compoundsA51 and A52 were found to completely retard tumor growth.

Other doses were also tried. At a daily dose of 1 mg/kg, compounds A51and A52 were found to have a mild effect. At a daily dose of 25-50mg/kg, compounds A51 and A52 were found induce some tumor cytotoxicity.

Prostate cancer cell lines were used for xenografts. For example, aLNCaP xenograft, LAPC4 xenograft, LAPC9 xenograft, and xenografts of thehormone refractory counterparts of these cell lines were made. Othercell lines included V-cap, CWR22 and LAPC4 cell lines. Two cell linesthat over express the androgen receptor were generated, LNCaP AR andLAPC4 AR. Prostate cancer progression in these engineered cell lines wasfound to differ from their parental counterparts. Under androgenablation, the LNCaP AR and LAPC4 AR lines continued to grow, thusbehaving like hormone refractory cells.

Some of the cell lines were found to not take well in mice in tumorformation when xenografted. However, with LNCaP, 2 million cells gave a95% take. As few as 1 million cells can be used. These cells required atleast 25% Matrigel but no more than 50%. Since high concentrations ofcells are required for good tumor take rate, a 27G needle was found tobe the smallest appropriate needle.

The LAPC4 cell line was found to be very difficult to grow in animals.The cells need to be resuspended and filtered through a micron meshfilter, for example, a 40-100 micron mesh filter, because theyfrequently form large aggregates. Resuspending and running through afilter helps normalize the cell number between each animal and thereforegives more consistent results. LAPC4 requires from about 25%-50%Matrigel, for example, 50% Matrigel, but can be grafted successfully ata lower concentration at 10⁵ cells.

Tumor take in SCID mice was found to be better than in nude mice. Forexample, the tumor take across individual animal in nude mice was foundto be very inconsistent. CB17 SCID mice were used in the study.

Injections were made subcutaneously on the right flank of the mouse.Slow injection was found to help to produce a round tumor that waseasier to measure and could be measured more accurately. In addition,because of the usage of Matrigel, injection of no more than 200 μl wasfound appropriate. Injection of 100-200 μl was found appropriate.Injecting too large a volume created leakage upon needle withdrawal.

An alternative method to help prevent leakage from needle pullout can beto warm the Matrigel:media:cells filled syringe a couple of seconds toproduce a gel-like form. When injecting the gel-like liquid, no leakageshould occur. However, allowing the Matrigel to heat for too long a timecan cause the suspension to solidify and become uninjectable.

PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION

The compounds of the invention are useful as pharmaceutical compositionsprepared with a therapeutically effective amount of a compound of theinvention, as defined herein, and a pharmaceutically acceptable carrieror diluent.

The compounds of the invention can be formulated as pharmaceuticalcompositions and administered to a subject in need of treatment, forexample a mammal, such as a human patient, in a variety of forms adaptedto the chosen route of administration, for example, orally, nasally,intraperitoneally, or parenterally, by intravenous, intramuscular,topical or subcutaneous routes, or by injection into tissue. Suchcompositions and preparations should contain at least 0.01% of acompound or compounds of the invention. The percentage of thecompositions and preparations may, of course, be varied and may, forexample, be between about 0.05% to about 2% of the weight of a givenunit dosage form. The amount of compounds in such therapeutically usefulcompositions is such that an effective dosage level will be obtained.

Thus, compounds of the invention may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier, or by inhalationor insufflation. They may be enclosed in hard or soft shell gelatincapsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the compounds may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.The compounds may be combined with a fine inert powdered carrier andinhaled by the subject or insufflated. Such compositions andpreparations should contain at least 0.1% of a compound or compounds ofthe invention. The percentage of the compositions and preparations may,of course, be varied and may conveniently be between about 2% to about60% of the weight of a given unit dosage form. The amount of compoundsin such therapeutically useful compositions is such that an effectivedosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch, orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid, and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose, or aspartame, or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac, sugar, and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye, and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the compounds of theinvention may be incorporated into sustained-release preparations anddevices. For example, the compounds may be incorporated into timerelease capsules, time release tablets, and time release pills.

The compounds of the invention may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the compoundscan be prepared in water, optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, triacetin, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations can contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the compounds of the invention which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid, and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions, orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers, or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the compoundsof the invention in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze drying techniques, which yielda powder of the active ingredient plus any additional desired,ingredient present in the previously sterile-filtered solutions.

For topical administration, the compounds of the invention may beapplied in pure form. However, it will generally be desirable toadminister them to the skin as compositions or formulations, incombination with a dermatologically acceptable carrier, which may be asolid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina, and the like. Other solidcarriers include nontoxic polymeric nanoparticles or microparticles.Useful liquid carriers include water, alcohols, or glycols orwater/alcohol/glycol blends, in which the compounds of the invention canbe dissolved or dispersed at effective levels, optionally with the aidof non-toxic surfactants. Adjuvants such as fragrances and additionalantimicrobial agents can be added to optimize the properties for a givenuse. The resultant liquid compositions can be applied from absorbentpads, used to impregnate bandages and other dressings, or sprayed ontothe affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses, or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of the present invention to the skin are known tothe art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392),Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157),and Wortzman (U.S. Pat. No. 4,820,508), all of which are herebyincorporated by reference.

Useful dosages of the compounds of Formula II can be determined bycomparing their in vitro activity, and by comparing their in vivoactivity in animal models. Methods for the extrapolation of effectivedosages in mice and other animals to humans are known to the art; forexample, see U.S. Pat. No. 4,938,949, which is hereby incorporated byreference.

For example, the concentration of the compounds in a liquid composition,such as a lotion, can be from about 0.1 to about 25% by weight, or fromabout 0.5 to about 10% by weight. The concentration in a semi-solid orsolid composition such as a gel or a powder can be from about 0.1 toabout 5% by weight, or from about 0.5 to about 2.5% by weight.

The amount of the compounds of the invention required for use intreatment will vary not only with the particular salt selected but alsowith the route of administration, the nature of the condition beingtreated and the age and condition of the patient and will be ultimatelyat the discretion of the attendant physician or clinician.

Effective dosages and routes of administration of agents of theinvention are conventional. The exact amount (effective dose) of theagent will vary from subject to subject, depending on, for example, thespecies, age, weight, and general or clinical condition of the subject,the severity or mechanism of any disorder being treated, the particularagent or vehicle used, the method and scheduling of administration, andthe like. A therapeutically effective dose can be determinedempirically, by conventional procedures known to those of skill in theart. See, e.g., The Pharmacological Basis of Therapeutics, Goodman andGilman, eds., Macmillan Publishing Co., New York. For example, aneffective dose can be estimated initially either in cell culture assaysor in suitable animal models. The animal model may also be used todetermine the appropriate concentration ranges and routes ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. A therapeutic dose canalso be selected by analogy to dosages for comparable therapeuticagents.

The particular mode of administration and the dosage regimen will beselected by the attending clinician, taking into account the particularsof the case (e.g., the subject, the disease, the disease state involved,and whether the treatment is prophylactic). Treatment may involve dailyor multi-daily doses of compound(s) over a period of a few days tomonths, or even years.

In general, however, a suitable dose will be in the range of from about0.01 to about 500 mg/kg per day, e.g., from about 0.1 to about 500 mg/kgof body weight per day, such as from about 0.1 to about 100 mg perkilogram body weight of the recipient per day. For example, a suitabledose may be about 1 mg/kg, 10 mg/kg, or 50 mg/kg of body weight per day.

The compounds of the invention are conveniently administered in unitdosage form; for example, containing from about 0.0005 to about 500 mg,from about 0.01 to about 50 mg, from about 0.05 to about 10 mg, or about5 mg of active ingredient per unit dosage form.

The compounds of the invention can be administered to achieve peakplasma concentrations of, for example, from about 0.5 to about 75 μM,about 1 to 50 μM, about 2 to about 30 μM, or about 5 to about 25 μM.Exemplary desirable plasma concentrations include at least or no morethan 0.25, 0.5, 1, 5, 10, 25, 50, 75, 100 or 200 μM. This may beachieved, for example, by the intravenous injection of a 0.05 to 5%solution of the compounds of the present invention, optionally insaline, or orally administered as a bolus containing about 1-1000 mg ofthe compounds. Desirable blood levels may be maintained by continuousinfusion to provide from about 0.0005 to about 25 mg per kg body weightper hour, for example at least or no more than 0.0005, 0.005, 0.05, 0.5,5, or 25 mg/kg/hr. Alternatively, such levels can be obtained byintermittent infusions containing from about 0.002 to about 100 mg perkg body weight, for example, at least or no more than 0.002, 0.02, 0.2,2, 20, 50, or 100 mg of the compounds per kg of body weight.

The compounds of the invention may conveniently be presented in a singledose or as divided doses administered at appropriate intervals, forexample, as two, three, four or more sub-doses per day. The sub-doseitself may be further divided, e.g., into a number of discrete looselyspaced administrations; such as multiple inhalations from aninsufflator.

Example Intravenous Formulation

A compound presently disclosed, for example, compound A51 or A52, can bein a formulation suitable for intravenous dosing. In an embodiment, thecompound is dissolved in from about 10% to about 25% dimethylsulfoxide(DMSO). IX phosphate buffered saline (PBS) is then mixed into thesolution as the balance, and the solution is sonicated with a water bathsonicator until it is homogeneous.

At a compound concentration of 1.5 mg/mL, 5 minutes of sonication may besufficient to dissolve the compound. At a compound concentration of 2mg/ml, more than 5 minutes of sonication may be required to dissolve thecompound and a polyethylene glycol can be added to keep the compound insuspension. For example, 5 to 40% PEG-400 (a polyethylene glycol), suchas, 5-10% PEG-400, can be added.

The above solution, including either A51 or A52, was found to be stableat room temperature for at least a week.

Before administration, the above solution should be sonicated for a fewminutes. A maximum appropriate administration volume for mice was foundto be 0.2 mL.

When administered to mice, hardening of the skin and skin irritationaround the injection site was observed, and this was attributed to theuse of DMSO. Although compounds A51 and A52 are soluble in ethanol,ethanol was found to reduce the stability of the compounds in vivo.

Over a period of 2 weeks following administration of the above solution,mice were observed to lose 15% of body weight.

Example Oral Formulation

A compound presently disclosed, for example, compound A51 or A52, can bein a formulation suitable for oral administration. In an embodiment, thecompound is dissolved in 100% DMSO.

Additional chemicals can be added, such as a carboxymethylcellulose, apolysorbate, or water. For example, the components of the solution otherthan A51 or A52 can be present at concentrations of from about 10% toabout 20% DMSO, from about 1% to about 2% carboxymethylcellulose (CMC),and 0.1% Tween 80 (a polysorbate), with the balance being water. Theconcentration of compound A51 or A52 in the oral foundation can be about1.5 mg/mL. The solution is mechanically homogenized for at least 30seconds. The compound A51 or A52 was found to stay in suspension foronly a couple of hours and, therefore, the oral formulation must beadministered within a couple of hours of preparation.

When more than 2% carboxymethylcellulose (CMC) was included in thesolution, the formulation was found to be very viscous, so that whenadministered to a test animal with a gavage syringe, much of theformulation was left behind on the walls of the syringe, preventingaccurate drug administration. A solution of 10% DMSO that included CMCand Tween 80 was found to keep the compound in suspension whenmechanical homogenization was applied. That is, more than 10% DMSO wasnot required. A minimum of DMSO should be used, because it was found toirritate the mice, and was associated with a loss of up to 10% of thebodyweight of the mice over a period of 2 weeks followingadministration.

A maximum appropriate administration volume for mice was found to be 0.2mL.

The half life of the compound was found to be longer when it wasadministered intravenously than when it was administered orally.However, daily oral dosing resulted in an acceptable steady state serumconcentration of the compound, comparable to the steady stateconcentration seen with bicalutamide. Oral administration may be moreconvenient than intravenous administration.

Compounds A51 and A52 have a beneficial effect on tumors in an in vivoassay administered as described.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Nothing in thisspecification should be considered as limiting the scope of the presentinvention. All examples presented are representative and non-limiting.The above-described embodiments of the invention may be modified orvaried, without departing from the invention, as appreciated by thoseskilled in the art in light of the above teachings. It is therefore tobe understood that, within the scope of the claims and theirequivalents, the invention may be practiced otherwise than asspecifically described.

1.-37. (canceled)
 38. A pharmaceutical composition comprising atherapeutically effective amount of a compound having a formula:

or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier, diluent, or adjuvant, wherein the pharmaceuticalcomposition is in a unit dose form of a capsule, tablet, or pill. 39.The pharmaceutical composition of claim 38, comprising at least onebinder, excipient, disintegrating agent, lubricant, sweetening agent, orflavoring agent.
 40. The pharmaceutical composition of claim 39,containing a binder, wherein the binder comprises gum tragacanth,acacia, corn starch, or gelatin.
 41. The pharmaceutical composition ofclaim 39, containing an excipient, wherein the excipient comprisesdicalcium phosphate.
 42. The pharmaceutical composition of claim 39,containing a disintegrating agent, wherein the disintegrating agentcomprises corn starch, potato starch, or alginic acid.
 43. Thepharmaceutical composition of claim 39, containing a lubricant, whereinthe lubricant comprises magnesium stearate.
 44. The pharmaceuticalcomposition of claim 39, containing a sweetening agent, wherein thesweetening agent comprises sucrose, fructose, lactose, or aspartame. 45.The pharmaceutical composition of claim 39, containing a flavoringagent, wherein the flavoring agent comprises peppermint, oil ofwintergreen, or cherry flavoring.
 46. The pharmaceutical composition ofclaim 39, wherein the compound is:


47. The pharmaceutical composition of claim 39, wherein the compound is:


48. The pharmaceutical composition of claim 38, wherein the tablet,pill, or capsule is coated with gelatin, wax, shellac, sugar, or acombination thereof.
 49. The pharmaceutical composition of claim 39,wherein the pharmaceutical composition is in the form of a pill ortablet.
 50. The pharmaceutical composition of claim 46, comprising afinely divided solid carrier selected from the group consisting of talc,clay, microcrystalline cellulose, silica, and alumina.
 51. Thepharmaceutical composition of claim 39, wherein the pharmaceuticalcomposition is in the form of a capsule.
 52. The pharmaceuticalcomposition of claim 39, comprising a pharmaceutically acceptable liquidcarrier.
 53. The pharmaceutical composition of claim 52, wherein thepharmaceutically acceptable liquid carrier comprises water, an alcohol,glycol, or water/alcohol/glycol blend.
 54. The pharmaceuticalcomposition of claim 52, wherein the pharmaceutically acceptable liquidcarrier comprises vegetable oil or a polyethylene glycol.
 55. Thepharmaceutical composition of claim 51, further comprising a syntheticpolymer, fatty acid, fatty acid salt, fatty acid ester, fatty alcohol,modified cellulose, or modified mineral material thickener.
 56. Thepharmaceutical composition of claim 38, wherein the unit dose formcontains the compound of formula:

in a range of from about 0.0005 to about 500 mg per unit dose form. 57.The pharmaceutical composition of claim 38, wherein the unit dose formcontains the compound of formula:

in a range of from about 0.01 to about 50 mg per unit dose form.
 58. Thepharmaceutical composition of claim 38, wherein the unit dose formcontains the compound of formula:

in a range of from about 0.05 to about 10 mg per unit dose form.